Cultivated mammalian cells are often used for production of recombinant polypeptides. Mammalian cell culture offers many advantages over non-mammalian systems, including, for example, proper protein folding, assembly and post-translational modification. However, there still exist challenges to improving productivity of large-scale mammalian cultures including, for example, challenges relating to growth level, cellular stress, and translation rate. In many industrial cell culture processes, cells are cultured at a high density in a large-scale bioreactor as a suspension and often the cells are proliferated beyond their optimal growth conditions. Under these conditions, apoptosis may be triggered, and as a result, cell viability and productivity may decrease. Consequently, many production optimization strategies rely on prevention of apoptosis and altering cellular metabolism by enhancing media formulations and growth conditions. Recent research suggests that cell productivity can be increased by altering global gene expression patterns of key molecules, such as transcription factors, that regulate multiple critical cellular pathways.
MicroRNAs (miRNAs) are small non-coding RNA molecules of about 22 nucleotides that are found in plants and animals and are key transcriptional and post-transcriptional regulators of gene expression. miRNAs function by base-pairing with complementary sequences within mRNA molecules, often resulting in gene silencing and are involved in diverse biological pathways in animals and plants including regulatory functions relating to cell growth, development and differentiation. miRNAs play a key role in maintaining cellular homeostasis and regulating important cellular pathways, such as growth and apoptosis. Inappropriate miRNA expression has been associated with a number of diseases, including cancer, where they may contribute to pathogenesis by altering numerous proteins and pathways simultaneously.
The ability for a change in a single miRNA to affect multiple physiological processes indicates that modifying miRNA expression in production cell culture may extend the productive cell growth phase, generate higher antibody titers and increase productivity (Sampson et al. (2007) “MicroRNA Let-7a down-regulates MYC and reverts MYC-induced growth in Burkitt Lymphoma cells.” Cancer Res 67(20):9762-9770; Muller et al. (2008) “MicroRNAs as targets for engineering of CHO cell factories.” Trends in Biotechnology 26(7):359-365; and Barron et al. (2011) “Engineering CHO cell growth and recombinant polypeptide productivity by over expression of miR-7.” Journal of Biotechnology 151(2):204-11). Accordingly, investigators have begun to examine the role of microRNAs in mammalian cell cultures, primarily through analysis of alterations in endogenous miRNAs that occur throughout production culture (Muller et al. (2008) “MicroRNAs as targets for engineering of CHO cell factories.” Trends in Biotechnology 26(7):359-365; Barron et al. (2011) “Engineering CHO cell growth and recombinant polypeptide productivity by over expression of miR-7.” Journal of Biotechnology 151(2):204-11; Gammell et al. (2007) “Initial identification of low temperature and culture stage induction of miRNA expression in suspension CHO-K1 cells.” Journal of Biotechnology 130:213-218; and Hackl et al. (2010) “Next-generation sequencing of the Chinese hamster ovary microRNA transcriptome: Identification, annotation and profiling of microRNAs as targets for cellular engineering.” Journal of Biotechnology 153(1-2):62-75). A small number of studies have also explored the effect of ectopically expressed miRs or anti-miRs on CHO cells (Barron et al., 2011; Meleady et al., 2011; Druz et al., 2011). However, more careful characterization of the effects of altered miRNA in CHO cells is necessary before this technology can be implemented routinely to increase production of therapeutic biologics.